Characterization of cuticular proteins in the red flour beetle, Tribolium castaneum

We are characterizing the cuticular proteins of Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae) to determine their role in the function of the exoskeleton. Based on qualitative analyses of cuticles, we focused on the sodium dodecyl sulfate (SDS)-extractable proteins. A small-scale cuticle "mini-prep" procedure was devised that yields preparations virtually free of contaminating cellular material compared to hand-dissected preparations, as assessed by fluorescent microscopy using DAPI to stain nuclei. Proteins extracted in 1% SDS from various developmental stages (last larval instar, pupal, adult) were analyzed by one-dimensional denaturing polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. The cuticular protein profiles show both similarities and differences among the stages examined. The amino acid composition, glycosylation, and partial amino acid sequence of several abundant cuticular proteins indicate similarity to cuticular proteins of other insects.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins

The cuticle is a biological composite material consisting principally of N‐acetylglucosamine polymer embedded in cuticular proteins (CPs). CPs have been studied and characterized by mass spectrometry in several cuticular structures and in many arthropods. Such analyses were carried out by protein extraction using SDS followed by electrophoresis, allowing detection and identification of numerous CPs. To build a repertoire of cuticular structures from Bombyx mori, Apis mellifera and Anopheles gambiae the use of SDS and electrophoresis was avoided. Using the combination of hexafluoroisopropanol and of a surfactant compatible with MS, a high number of CPs was identified in An. gambiae wings, legs and antennae, and in the thoracic integument cuticle of Ap. mellifera pupae. The exoskeleton analysis of B. mori larvae allowed to identify 85 CPs from a single larva. Finally, the novel proteomics approach was tested on cuticles left behind after the molt from the fourth instar of Acyrthosiphon pisum. Analysis of these cast cuticles allowed to identify 100 Ac. pisum CPs as authentic cuticle constituents. These correspond to 68% of the total putative CPs previously annotated for this pea aphid. While this paper analyzes only the recovered cuticular proteins, peptides from many other proteins were also detected.     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

Cuticular proteins: The neglected component

This paper emphasizes the importance of the protein component of cuticles. Correlation of electrophoretic charge distribution of individual cuticular proteins and physical properties of the cuticles from which they were extracted, as well as interpopulation and interspecies conservation of electrophoretic patterns, are used to argue that individual proteins play precise roles in the cuticle. Glycosylation of cuticular proteins is described, but no function for these modifications is yet known. Analogy is drawn to analyses of chorion proteins and the case is made that analysis of amino acid sequence data is likely to provide insights into how cuticular proteins and chitin interact to construct the diverse types of cuticles.     

Expanding abdominal cuticle in the bug Rhodnius and the tick Boophilus

The abdominal cuticles of Rhodnius prolixus (fifth instar) and Boophilus microplus (adult female) expand dramatically and rapidly during feeding. In the unfed stage of both species the epicuticle of the abdomen is deeply folded, and when rapid stretching takes place the epicuticle unfolds and the underlying procuticle stretches so that the thickness of the cuticle is halved. The cuticles contained only trace amounts of protein soluble in water and aqueous KCl but substantial quantities were extracted with 7 M aqueous urea. The proteins were analysed for their amino acid composition and investigated by gel electrophoresis and isoelectric focusing.In solubility, amino acid composition, molecular weight distribution, and isoelectric points, the proteins isolated from both species resembled one another closely. They had higher molecular weights and higher isoelectric points than did the proteins from flexible, non-stretching cuticles and unlike them had high alanine and histidine and low aspartic acid and glutamic acid contents. Their amino acid composition was very similar to that of the whole cuticle. The proteins were not associated with neutral sugars. Both the Rhodnius and Boophilus cuticles had low chitin contents, 11·2 and 3·8% respectively (on a water-free basis). The composition of the cuticles and the properties of the proteins are discussed in relation to the stretching which they undergo.     

Pupal and larval cuticle proteins of Drosophila melanogaster

Proteins, soluble in 7 M urea, were extracted from third-instar larval and pupal cuticles of Drosophila melanogaster. Both extracts contain a limited number of polypeptides resolved by one- or two-dimensional electrophoresis. The five major larval proteins have low molecular weights (less than 20000) and are not glycosylated. The major pupal cuticle proteins fall into two size classes: two with apparent molecular weights of 56K and 82K and four with molecular weights between 15K and 25K. The proteins with high apparent molecular weights are glycosylated. In nondenaturing gels, no components of the larval and pupal cuticle extracts comigrate. One-dimensional "fingerprints" indicate that cuticle proteins from these two stages have unique primary structures. Immunological results indicate that the major low molecular weight larval and pupal cuticle proteins are comprised of two families of proteins that share antigenic determinants. The high molecular weight pupal cuticle proteins are immunologically unrelated to the low molecular weight components. We conclude that the pupal and larval proteins are encoded in part by multigene families that have arisen by gene duplication and evolutionary divergence.     

The cuticular proteins from free-living and parasitic stages of Haemonchus contortus--I. Isolation and partial characterization

1. Cuticles were isolated from the adult males, adult females, the second molt (2M) sheath from the infective larvae (L3(2M)), and the parasitic third stage (L3) of the sheep parasite Haemonchus contortus by a combination of mechanical disruption and detergent treatment. 2. The 2ME soluble cuticular proteins from adult males contained 4 or 5 major protein bands with molecular weights ranging from 100 to 56 kD with the most prominent band at 56 kD. The cuticular proteins from adult females were similar to the male. 3. Cuticular proteins from the larval stages, 2M cuticle, and L3 cuticle, differed from the adults and from each other. The most prominent protein bands were observed with molecular weights on 78 and 39 kD for the L3 cuticle and 100, 91 and 46 kD for the 2M cuticle. The 2ME soluble cuticular proteins from all developmental stages were at least partially digested by bacterial collagenase. 4. The amino acid composition of cuticular proteins was similar for the L3 and 2M, but adults had lesser amounts of glycine and greater amounts of basic amino acids than the larval stages. The amount of the isolated cuticle solubilized by the 2ME treatment was greatest in adults (80%) compared to the L3 (64%) and the 2M (22%). 5. These results support a hypothesis that there are quantitative and qualitative stage specific differences in the cuticular proteins of H. contortus.     

Developmental changes in cuticular proteins of Ascaris suum

1. Cuticles were isolated from developmental stages of the swine nematode Ascaris suum by a combination of mechanical disruption and detergent treatment of larvae or by surgical removal of cuticle from adults. Proteins from the isolated cuticles were solubilized with 2-mercaptoethanol (2ME) and analyzed by SDS-PAGE. 2. 2ME soluble, cuticular proteins from adults consisted of 5 to 6 bands with 80% of proteins in 2 bands with mol. wts of 106,000 and 93,000. Cuticular proteins from the third and fourth larval stages (L3 and L4) were comparable to adult, but differences in the number of bands were observed. The soluble proteins from the adult, L3 and L4 were readily degraded by a bacterial collagenase suggesting that these proteins are collagen-like structural elements of the cuticle. 3. The soluble proteins from the second stage (L2) differed from the adult and other larval stages in both the number and mol. wt of protein bands and their lack of degradation by bacterial collagenase. Amino acid composition of soluble cuticular proteins were similar for adult and L4, but glycine and proline were present in lower amounts in the L2. 4. These results support a hypothesis that there are stage specific differences in cuticular proteins from A. suum and that the greatest differences appear to exist between L2 and other stages.     

The cuticular proteins of Tenebrio molitor: II. Patterns of synthesis during postembryonic development

The program of synthesis for the soluble cuticular proteins of Tenebrio molitor was determined by following the incorporation of labeled leucine after a 4-hr pulse in vivo. Soluble proteins were extracted from labeled cuticles and separated on SDS-polyacrylamide slab gels; individual gel slices were counted. The synthetic patterns of larvae and pupae were similar to one another but distinct from the adult pattern. At each stage, the preecdysial pattern was unlike that of postecdysial animals. Distinct periods of synthesis were detected for different proteins. One protein was synthesized and deposited throughout cuticle formation in all three metamorphic stages. One group was synthesized only after ecdysis, while synthesis and secretion of other proteins were restricted to the preecdysial period. Some cuticular proteins never acquired detectable label.     

Correlated changes in mechanical properties of the intersegmental membrane and bonding between proteins in the female adult locust

When the female locust ecdyses into the adult instar the intersegmental membranes which will extend when the female digs the oviposition hole are stiff and inextensible. Extensibility of the membranes develops later, and is under the control of the corpus allatum: allatectomy prevents the development of extensibility. The difference in extensibility and stiffness of the membranes before and after sexual maturation can be accounted for as a change in the bonding in the protein matrix of the cuticle. Electrophoretic analysis of the cuticular proteins dissociated by SDS shows that there are five major proteins in the cuticle, and that these do not change when the membrane becomes extensible. The differing responses of the two states of the cuticle to sequential extraction in water, 0·5 M KCl, 7 M urea, and 1·0 M NaOH at 60°C suggest that the change is one of reduction in hydrogen bonding. Simple calculations on mechanical data suggest that in the unstretchable state there is one interchain bond every 370 or so amino acid residues. This may allow sufficiently specific site definition for an enzyme to be involved in the breakdown of hydrogen bonding. An E.M. of a possible enzyme package in the cuticle is presented.     

Purification of phenolic compounds and a phenoloxidase from larval cuticle of the red-humped oakworm,Symmerista cannicosta francl

A phenoloxidase has been extracted, purified, and characterized from cuticle of last-instar larvae of the red-humped oakworm, Symmerista cannicosta. It is a typical tyrosinase (EC 1.10.3.1., o-diphenol:O₂ oxidoreductase), active toward o-diphenols but not p-diphenols, inhibited by thiourea and phenylthiourea, with a pH optimum between 6.0 and 7.2. In these respects it resembles enzyme A of C. vicina, one of the few species from which this presumed wound healing enzyme has been purified and characterized. Hydrolysis of either exuviae or intact cuticle from last instar larvae yielded a number of ketocatechols of which the most abundant, 2-hydroxy, 3′,4′-dihydroxyacetophenone, represented 2.9% of the dry weight of head capsule exuviae, 0.3% of exuviae from the remainder of the body, and 4.6% of the dry weight of head capsule cuticle from previously frozen intact larvae. Differences in the type and amount of ketocatechol recovered from these cuticles are described.     

Consideration of the importance of hydrophobic interactions in stabilizing insect cuticle

Protein fractions of insect cuticles with different mechanical properties have related values of polarity and hydrophobicity. Hydrophobicity is important for the self-assembly of cuticle which is produced prior to the moult and in plasticization of cuticle. The cuticles of soft-bodied fly larvae are quite distinct from those of exopterygotes (e.g. locusts) and this can be related to the chemistry and mode of tanning. The properties of cuticular proteins are compared: the proteins of the pliant cuticles most closely resemble globulins, and the proteins in stiff cuticles are more like fibrous and hydrophobic structural proteins. Changes in the environment of the proteins may alter their shape and hence the mechanical properties of the cuticle.     

Analysis of the cuticular proteins of Hyalophora cecropia with two dimensional electrophoresis

The soluble cuticular proteins of defined anatomical regions from different metamorphic stages of the giant silkmoth, Hyalophora cecropia, were characterized by two dimensional electrophoresis. As urea concentrations in 2D gels were increased, some of the cuticular proteins from the larval dorsal abdomen decreased in mobility relative to the molecular weight standards. This decrease was also influenced by the pH and ionic strength of the resolving gel. Clustering of proteins into groups, whose members showed similar behavior under different electrophoretic conditions, was indicative of membership in multigene families. By such criteria, common families were found in cuticles with similar mechanical properties from different metamorphic stages, yet there was evidence that different members of a single family were independently regulated.     

The isolation and immunogenicity of the cuticle of Dipetalonema viteae (Filarioidea)

The cuticle of the filaria Dipetalonema viteae was isolated by sonication in 1% sodiumdodecylsulphate (SDS) and in a mixture of 1% SDS and 5% B-mercaptoethanol (BME). Sonication in SDS removed all internal parts and left the cuticle intact; this was verified by light- and electronmicroscopy. Sonication and incubation of the cuticle in the mixture of SDS-BME at pH 6.8 dissolved the basal and part of the median zone of the cuticle. The epicuticle and the cortical zone remained intact. The extracts were investigated using SDS-polyacrylamide gel electrophoresis; the early extracts contained a wide variety of proteins, whereas the later steps showed a consistent pattern with a smaller number of bands. Cuticles after SDS-purification, the extract of cuticular material in SDS-BME, and the cuticles insoluble in SDS-BME were used to immunize mice; the antibodies produced were visualized by an indirect fluorescent antibody test on cryostat sections of female worms. When SDS-purified cuticles were used for immunization, antibodies directed against all organs in the filariae were found. The SDS-BME extract and the insoluble cuticular pellet stimulated the production of antibodies restricted to the cuticle of adult worms and microfilariae. The purification method opens up the possibility of further isolation and characterization of antigens from the cuticle.     

The effects of DOPA decarboxylase inhibitors on the permeability and ultrastructure of the larval cuticle of the Australian sheep blowfly, Lucilia cuprina

Larvae of Lucilia cuprina, fed toxic levels of α-methyl DOPA (or other DOPA decarboxylase inhibitors) during the first or second instar, die at the completion of the next moult, soon after exposing their new cuticles. In electron micrographs of newly synthesised cuticle from these treated larvae, the ultrastructure of the lipid-rich outer epicuticle layer appears to be abnormal. This newly formed cuticle of the treated larvae is apparently defective in its role as a water permeability barrier (compared with that of normal larvae), since it permits the free movement of water in both directions. Thus, treated larvae die most probably as a direct result of dehydration. Larvae fed toxic levels of α-methyl DOPA can be rescued from death by simultaneously adding N-acetyldopamine (the cuticular sclerotizing agent) to the food. The rescued larvae are apparently normal in all respects. This suggests that sclerotization is required for the formation of a normal outer epicuticle. Diflubenzuron, which is known to inhibit chitin deposition in the cuticles of a number of different species of insect, also apparently affects chitin deposition in the larval cuticle of L. cuprina. Thus, in electron micrographs of cuticle from larvae fed toxic levels of diflubenzuron the ultrastructure of the chitin-containing endocuticle layer appears to be abnormal.     

Tyrosine-derived cross-linking amino acids in the sheath of Haemonchus contortus infective larvae

The sheath or second-molt cuticle (2M) was isolated from in vitro exsheathed Haemonchus contortus infective larvae (L3[2M]). Acid hydrolysates of 2-mercaptoethanol (2ME)-soluble and 2ME-insoluble cuticular proteins were analyzed by high performance liquid chromatography for tyrosine-derived cross-linking amino acids. Dityrosine and isotrityrosine were identified by their chromatographic behavior, absorbance spectra, and other chemical characteristics in both the 2ME-soluble and 2ME-insoluble fractions. Dityrosine and isotrityrosine were found in greater amounts in the 2ME-insoluble proteins. When intact 2M cuticles were labeled with 125I prior to acid hydrolysis, radiolabel was recovered in tyrosine but not dityrosine or isotrityrosine indicating that the tyrosine cross-links are not susceptible to iodination in the intact protein. The results are consistent with a hypothesis that tyrosine-derived cross-links are important components of H. contortus 2M cuticular proteins.     

Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Post-translational modifications are a conspicuous feature of the proteins of vertebrate extracellular matrices such as cartilage. Yet this feature remains virtually unexplored with insect cuticle, a situation this paper begins to remedy. Cuticular proteins were extracted from cuticles of Hyalophora cecropia and separated on isoelectrofocusing and 2D gels. Periodic acid-Schiff reagent stained several proteins from flexible cuticles and a few proteins from rigid cuticles, indicating that some proteins were glycosylated. Elucidation of the specific nature of this glycosylation came from probing electrophoretically separated cuticular proteins blotted onto nitrocellulose with biotinylated lectins. Most major cuticular proteins did not react; minor cuticular proteins and molecules which do not stain with Coomassie blue were found to bind lectins specific for mannose and N-acetylgalactosamine. Limited binding was also detected with lectins specific for N-acetylglucosamine, galactose and fucose. No sialic acid was detected using either lectins or neuraminidase digestion. The amount of glycosylation was greatest in proteins extracted from flexible cuticles. Although several proteins stained with Alcian blue indicating presence of sulfation, ³⁵S which had been incorporated at low levels in cuticular proteins corresponded to [³⁵S]methionine. No indication of the presence of mammalian-type glycosaminoglycans in insect cuticles was obtained after treatment with chondroitinase or nitrous acid. The functional significance of the modifications detected remains unknown. No evidence for phosphorylated proteins or lipoproteins was found.     

Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm

The pattern of cuticular protein synthesis by the epidermis of the tobacco hornworm larva changes during the final day of feeding, leading to an alteration in cuticular structure and a stiffening of the cuticle. We have isolated a small multigene family which codes for at least three of the new cuticular proteins made at this time. The five genes which were isolated from this family map to two different genomic regions. Sequencing shows that one of the genes is 1.9 kb and consists of three exons coding for a 12.2-kDa acidic (pI = 5.26) protein that is predominantly hydrophilic. The deduced amino acid sequence shows regions of similarity to proteins from flexible lepidopteran cuticles and from Drosophila larval and pupal cuticles, but not to proteins found in highly sclerotized cuticles. This gene family is first expressed late on the penultimate day (Day 2) of feeding in the final larval instar and ceases expression 2 days later when metamorphosis begins. In situ hybridization shows that this gene family is expressed in all the epidermal cells of Day 3 larvae except the bristle cells and those at the muscle attachment site. Expression can be induced in Day 1 epidermis by exposure to 50 ng/ml 20-hydroxyecdysone in vitro, but only if juvenile hormone is absent. Its developmental expression, tissue specificity, and hormonal regulation strongly suggest that this multigene family is involved in the structural changes that occur in the larval cuticle just prior to the onset of metamorphosis.